The hormonal factors which stimulate the biosynthesis of proteoglycans by rat ovarian granulosa cells in vitro have been examined in detail. LH, FSH, and HCG have been shown to stimulate proteoglycan synthesis. The identity of these proteoglycans has been determined, and the dose response curves have been characterized. In addition, the effects of estradiol, testosterone, various prostaglandins, and cyclic AMP have been studied in this system. The results provide a necessary background for further studies of the physiology of biosynthesis of ovarian proteoglycans in normal and abnormal states.